An increasing body of evidence suggests that the complement system regulatory glycoprotein, factor H (FH), if produced in sufficient quantities and endowed with appropriate pharmacokinetic and pharmacodynamic properties, would serve as a new biotherapeutic agent. This agent could prevent development of age-related macular degeneration (AMD) in genetically susceptible individuals and facilitate treatment in those with AMD and two life-threatening kidney conditions known as atypical haemolytic uraemic syndrome (aHUS) and dense deposit disease (DDD). More speculatively, this agent could have beneficial effects in the treatment or prevention of numerous other diseases in which inadequate complement regulation contributes to aetiology or symptoms.
However current attempts to produce FH through over-expression of a gene in recombinant cells have failed to yield the quantities that would be required for therapy, while purification from human plasma of sufficient quantities of the appropriate variants of FH has logistical and technical difficulties and carries health risks. There are urgent unmet clinical and commercial needs for multiple-gram quantities of biotherapeutic-grade recombinant versions of FH with minimal immunogenicity, an extended half-life and maximal efficacy.
Links between polymorphisms in FH and susceptibility to disease have been well documented and are reviewed, for example in: Opportunities for new therapies based on the natural regulators of complement activation. Brook E, Herbert A P, Jenkins H T, Soares D C, Barlow P N. Ann NY Acad Sci 2005 1056:176-88; Complement factor H: using atomic resolution structure to illuminate disease mechanisms. Barlow P N, Hageman G S, Lea S M. Adv Exp Med Biol. 2008 632:117-42; Translational mini-review series on complement factor H: renal diseases associated with complement factor H: novel insights from humans and animals. Pickering M C, Cook H T. Clin Exp Immunol 2008 151:210-30; Translational mini-review series on complement factor H: genetics and disease associations of human complement factor H. de Córdoba S R, de Jorge E G. Clin Exp Immunol. 2008 151:1-13.
Since these reviews were published numerous further published findings have broadened the scope of potential targets for FH-based therapies. Two recent examples establish an association between the FH gene (CFH) polymorphism (Y402H) and susceptibility to cardiovascular disease (CVD). Koeijvoets et al. (Complement factor H Y402H decreases cardiovascular disease risk in patients with familial hypercholesterolaemia. Koeijvoets K C, Mooijaart S P, Dallinga-Thie G M, Defesche J C, Steyerberg E W, Westendorp R G, Kastelein J J, van Hagen P M, Sijbrands E J. Eur Heart J. 2009 30:618-23. showed that amongst patients with severely increased risk of early-onset CVD due to hypercholestrolaemia, the Y402 CFH variant was inversely associated with susceptibility to CVD suggesting that CFH modifies the risk of CVD. In a study by Buraczynska et al. (Complement factor H gene polymorphism and risk of cardiovascular disease in end-stage renal disease patients. Buraczynska M, Ksiazek P, Zukowski P, Benedyk-Lorens E, Orlowska-Kowalik G. Clin Immunol. 2009; 132:285-90) of end-stage renal failure in patients on dialysis, multivariate logistic regression analysis showed that the Y402H genotype is independently associated with cardiovascular co-morbidity; with homozygosity for the H402 allele being associated with an odds ratio of 7.28 (95% CI 5.32-9.95). In another recent development, Moreno-Navarrete et al. (Complement Factor H is expressed in adipose tissue in association with insulin resistance. Moreno-Navarrete J M, Martínez-Barricarte R, Catalán V, Sabater M, Gómez-Ambrosi J, Ortega F J, Ricart W, Blüher M, Frühbeck G, de Cordoba S R, Fernández-Real M J. Diabetes 2009: Epub Oct. 15) showed that FH is expressed in adipose tissue in association with insulin resistance, suggesting a link between the alternative pathway of the complement system, obesity and metabolic disorders.
Data for the likely efficacy of FH in treatment is already very strong, and has precipitated numerous disclosures, patent applications and company start-ups. US2007/0020647 discusses the expression of human CFH in a variety of eukaryotic and prokaryotic protein-overproduction vectors and in mammalian cell lines, but only explicitly exemplifies expression in the human lung carcinoma cell line A549. The quantities of recombinant protein obtained from this cell line are not disclosed, but based on precedent and in the absence of any evidence to the contrary the amounts are expected to be inadequate for therapeutic purposes. WO2007/038995 describes the use of human factor H to treat aHUS. The patent application mentions the use of recombinant FH without providing significant details about the methods of production of recombinant FH, but is focused on purification of FH from human plasma.
Thus although the above two documents disclose the idea of using recombinant FH therapeutically, neither document actually teaches the large-scale production of recombinant FH that is absolutely essential for its therapeutic application; as shown herein, this is not a straightforward task.
Successful manufacture of larger amounts (greater than 10 mg) of pure recombinant full-length FH with preserved functional activities has not previously been reported in the scientific or patent literature. Indeed, in the limited data supporting the patents discussed above, the authors demonstrated capability of producing only minute quantities (less than about 1 mg) of recombinant FH and did not provide evidence that they had purified or characterised this material. Furthermore, literature reports likewise allude to sub-milligram quantities of recombinant FH from insect and mammalian cells (e.g. Biologically active recombinant human complement factor H: synthesis and secretion by the baculovirus system. Sharma A K, Pangburn M K. Gene 1994 143:301-2; Structural and functional characterization of factor H mutations associated with atypical hemolytic uremic syndrome. Sánchez-Corral P, Pérez-Caballero D, Huarte O, Simckes A M, Goicoechea E, López-Trascasa M, de Córdoba S R. Am J Hum Genet 2002 71:1285-95.) or to expression of fragments, only, of the FH molecule (e.g. Structure of the N-terminal region of complement factor H and conformational implications of disease-linked sequence variations. Hocking H G, Herbert A P, Kavanagh D, Soares D C, Ferreira V P, Pangburn M K, Uhrin D, Barlow P N. J Biol Chem 2008 283:9475-87).
Ormsby, R. J. et al., Expression of human factor H in the methylotrophic yeast Pichia Pastoris. Molecular Immunology Vol 35, p. 353, 1998 Abstract 92. This paper uses a Pichia pastoris production system to express a FIVE (5) complement control protein (CCP) fragment of Factor H, not the full length TWENTY (20) CCP Factor H protein, which is the subject of present patent application.
Ripoche, J. et al., The complete amino acid sequence of human complement Factor H. Biochemical Journal, Vol 249: 593-602, 1988. This paper describes the full length human factor H nucleotide sequence (and hence the amino acid sequence) and was obtained by sequencing three overlapping cDNA clones spanning the Factor H gene. However, it does not describe how to clone the gene such that it is possible to express functional human Factor H protein.
EP1336618 describes using full length or fragments of porcine Factor H as a soluble complement regulator, for use as a therapeutic. It is suggested that porcine factor H could be purified from pig plasma or as exemplified in this patent, made recombinantly using Baculovirus. However, no quantification of the amount of full length porcine factor H from a standard fermentation nor any functional data for the full length protein (rather than only fragments) is shown. However, there is no disclosure or teaching of how to express functional human Factor H.
The use of porcine Factor H naturally carries the risk of infection with cross-species zoonotic infections. Moreover, there is not complete DNA sequence or amino acid homology between human factor H and porcine factor H (62% homology Hegasy G. A. et al., Pig complement regulator factor H: molecular cloning and functional characterization. Immunogenetics. 2003 October; 55(7):462-71). It is therefore very likely autoantibodies to porcine Factor H would be made, which would again limit therapeutic usage.
WO 2008/135237 describes use of a therapeutic which combines a short consensus repeat (SCR) of Factor H with a pathogen recognition binding molecule e.g. an antibody. It specifically mentions use of fragments/peptide chains of less than 100 amino acids (<2 SCRs). It does not suggest use of a full length Factor H molecule with a pathogen recognition binding molecule. Also, its focus is for the use of treating infections or for cancer, not renal or opthalmological diseases.
Currently, FH-replacement clinical therapy is achieved by means of infusing donated pooled plasma, of which FH is only one of many protein components. It is not possible clinically to routinely obtain plasma containing only the FH Y402 allotype (which is protective against AMD); when purified in bulk from pooled plasma, FH is heterogeneous in terms of both its heterotypic and glycoform variations and hence this material is ill-suited for therapy; antibody-affinity based purification methods generally yield only small amounts (a few mg at most) of material that can be enriched only for a single variant at a specific site of variation (e.g. for Y402) but will be heterogeneous with respect to other polymorphic sites (e.g. V62I). Any use of plasma-purified human proteins would in any case may carry unacceptable risks, of infection with both unknown viral and prion proteins, and of sensitisation to contaminating plasma components, when used on the repetitive basis proposed for AMD, aHUS and DDD therapies.
It is therefore amongst the objectives of the present invention to obviate and/or mitigate at least one of the aforementioned obstacles to therapeutic use of FH.